本文關(guān)鍵詞：紫外誘導(dǎo)圓錐鐵線蓮中吲哚生物堿合成機(jī)理研究及CtIPT的克隆與原核表達(dá)分析　出處：《浙江大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 圓錐鐵線蓮 UV-B誘導(dǎo) 吲哚生物堿 代謝組學(xué) 蛋白質(zhì)組學(xué) 腺苷酸異戊烯基轉(zhuǎn)移酶 克隆與原核表達(dá)

【摘要】：中國是藥用植物資源最豐富的國家之一,而藥用植物在人類疾病預(yù)防和治療中占有重要地位。UV-B對藥用植物的生長發(fā)育和次生代謝影響很大,但是,不同藥用植物對UV-B的具體響應(yīng)情況有所不同,許多機(jī)理有待進(jìn)一步發(fā)現(xiàn)。吲哚生物堿是藥用植物中非常具有研究價(jià)值的一類次生代謝產(chǎn)物,在UV-B逆境下含量增加,不僅能夠增強(qiáng)植物自我保護(hù)能力以抵御逆境脅迫,還具有細(xì)胞毒性、抗癌、抗病毒、抗瘧和抗炎等功效,能夠直接增加植物的藥用價(jià)值。細(xì)胞分裂素是由腺苷酸異戊烯基轉(zhuǎn)移酶合成的一類植物激素,在UV-B逆境下含量減少,不僅能夠調(diào)控藥用植物生長發(fā)育使其形態(tài)學(xué)發(fā)生改變而處于抵御逆境脅迫的狀態(tài),還可能與UVR8信號通路中的某些調(diào)控因子相關(guān),參與調(diào)控植物次生代謝產(chǎn)物的合成(如吲哚生物堿、香豆素、黃酮等),能夠間接增加植物的藥用價(jià)值。圓錐鐵線蓮屬于毛茛科,具有非常高的藥用價(jià)值。實(shí)驗(yàn)室前期從圓錐鐵線蓮中分離出了吲哚生物堿(6-hydroxyl-1H-indol-3-yl)carboxylic acid methyl ester,本課題在該研究成果的基礎(chǔ)上進(jìn)行了更深入的研究,主要研究內(nèi)容和結(jié)果如下:(1)圓錐鐵線蓮中吲哚生物堿響應(yīng)UV-B誘導(dǎo)的合成機(jī)理HPLC-TOF-MS/MS分析顯示圓錐鐵線蓮經(jīng)過UV-B誘導(dǎo)后其吲哚生物堿(6-hydroxyl-1H-indol-3-yl)carboxylic acid methyl ester 含量增加,表明圓錐鐵線蓮在UV-B誘導(dǎo)下體內(nèi)吲哚生物堿合成途徑呈增強(qiáng)狀態(tài)�；�2-DE和GC-TOF-MS技術(shù)的蛋白質(zhì)組學(xué)和代謝組學(xué)研究揭示了圓錐鐵線蓮經(jīng)UV-B誘導(dǎo)后吲哚生物堿的合成機(jī)理,即與氨基酸代謝相關(guān)的蛋白和化合物含量顯著增加,表明氨基酸代謝被激活。通過對絲氨酸脫氨酶進(jìn)行酶活檢測,結(jié)果顯示該酶活經(jīng)過UV-B誘導(dǎo)后顯著增強(qiáng),表明氨基酸代謝過程被UV-B輻射激活能夠促進(jìn)下游莽草酸代謝途徑的增強(qiáng)�；趒RT-PCR技術(shù)的轉(zhuǎn)錄組學(xué)研究分析了圓錐鐵線蓮經(jīng)UV-B誘導(dǎo)后吲哚生物堿合成途徑上關(guān)鍵基因的變化情況,結(jié)果顯示從莽草酸到色氨酸代謝過程中的8個(gè)關(guān)鍵基因的表達(dá)表現(xiàn)出上調(diào)共性;通過HPLC-TOF-MS/MS代謝組檢測圓錐鐵線蓮經(jīng)UV-B誘導(dǎo)后吲哚生物堿合成途徑上關(guān)鍵化合物的變化情況,結(jié)果顯示鄰氨基苯甲酸鹽、吲哚和色氨酸的含量都增加;通過對色氨酸合成酶進(jìn)行酶活檢測,結(jié)果顯示該酶活經(jīng)過UV-B誘導(dǎo)后顯著增強(qiáng),上述結(jié)果均表明增強(qiáng)了的莽草酸代謝途徑能夠促進(jìn)吲哚生物合成途徑的增強(qiáng),最終為吲哚生物堿的合成奠定了基礎(chǔ)。(2)圓錐鐵線蓮中腺苷酸異戊烯基轉(zhuǎn)移酶的克隆與原核表達(dá)借助轉(zhuǎn)錄組測序,我們獲得了圓錐鐵線蓮葉片中腺苷酸異戊烯基轉(zhuǎn)移酶的EST片段,之后利用RT-PCR和cDNA末端快速擴(kuò)增技術(shù)(rapid amplification of cDNAends,RACE),得到了長度為1374 bp的堿基序列,其中包括長度為332 aa的完整ORF區(qū)。接著對該序列編碼的蛋白CtIPT進(jìn)行理化性質(zhì)預(yù)測、序列比對和系統(tǒng)進(jìn)化樹分析,結(jié)果顯示該蛋白是一個(gè)分子量為37.2 kDa的親水性蛋白,存在腺苷酸異戊烯基轉(zhuǎn)移酶特征序列區(qū)域(ATP/GTP結(jié)合序列區(qū)域),且與番茄中的S1IPT3/4和擬南芥中的AtIPT3/5/7非常相似,表明該蛋白為腺苷酸異戊烯基轉(zhuǎn)移酶。然后通過qRT-PCR檢測UV-B誘導(dǎo)對CtIPT表達(dá)的影響,結(jié)果顯示具有顯著性抑制作用,表明CtIPT可能具有抵御紫外逆境脅迫的功能。再分別以pET-28a(+)、pGEX-4T-1和pMAL-c2X質(zhì)粒為表達(dá)載體對CtIPT進(jìn)行克隆,并在大腸桿菌BL21中進(jìn)行原核表達(dá),SDS-PAGE結(jié)果顯示在16℃,180rpm,0.5mM IPTG誘導(dǎo)表達(dá)6 h的條件下均可以成功表達(dá)出目的蛋白,但是只有在BL21-pMAL-c2X-CtIPT(含MBP標(biāo)簽)中實(shí)現(xiàn)了可溶性表達(dá)。最后通過Amylose resin親和層析柱進(jìn)行分離純化,得到了目的蛋白腺苷酸異戊烯基轉(zhuǎn)移酶。
[Abstract]:Abstract: China is one of the most abundant resources of medicinal plants, and medicinal plants play an important role in the prevention and treatment of human diseases. UV-B has great influence on the growth and secondary metabolism of medicinal plants. However, the response of different medicinal plants to UV-B is different. Many mechanisms need to be further discovered. Indole alkaloids are a class of secondary metabolites is extremely valuable in medicinal plants, increase the content of UV-B in the face of adversity, not only can enhance the ability of self - protection against plant stress, also has cytotoxicity, anticancer, antiviral, antimalarial and anti-inflammatory effect, can directly increase the medicinal value of plants. Cytokinin is a kind of plant hormone synthesis by enzyme adenylate isopentenyl transferase, reduce the content of UV-B in the face of adversity, not only can adjust the growth and development of medicinal plants in the morphological changes in the resist stress state may be associated with some regulatory factors in the UVR8 signaling pathway, is involved in the regulation of synthesis of plant secondary metabolites the (such as Huang Tong, indole alkaloids coumarins, etc.), can indirectly increase the medicinal value of plants. Clematis Clematis is a species of Ranunculus, which has very high medicinal value. From the previous sweetautumn Clematis isolated indole alkaloids (6-hydroxyl-1H-indol-3-yl) carboxylic acid methyl ester, makes a further study of this topic based on the research results, the main research contents and results are as follows: (1) sweetautumn Clematis indole alkaloids in response to the HPLC-TOF-MS/MS synthesis mechanism induced by UV-B analysis showed that the cone of Clematis after the induction of UV-B (6-hydroxyl-1H-indol-3-yl) carboxylic indole alkaloids increased acid methyl ester content showed that sweetautumn Clematis induced by UV-B in vivo indole alkaloids biosynthesis pathway was to strengthen the state. Proteomics and metabonomics based on 2-DE and GC-TOF-MS technology revealed the mechanism of indole alkaloid synthesis after Clematis induction by UV-B, that is, the content of protein and compounds related to amino acid metabolism increased significantly, indicating that amino acid metabolism was activated. Through enzyme activity detection of serine deaminase, the results showed that the enzyme activity was significantly enhanced after UV-B induction, indicating that amino acid metabolism activated by UV-B radiation can promote the enhancement of downstream shikimic acid metabolism pathway. Transcriptome qRT-PCR technology research has analyzed the changes of key genes of sweetautumn Clematis induced by UV-B Indole Alkaloids Biosynthesis Pathway Based on the results showed that the expression of 8 key genes from shikimic acid to tryptophan metabolism in the process of showing up in common; changes of key compounds through the HPLC-TOF-MS/MS metabolic detection of sweetautumn Clematis after induced by UV-B indole alkaloid biosynthesis pathway. Results showed that the anthranilic acid salt, indole and tryptophan content were increased; the tryptophan synthase enzyme activity assay, the results show that the enzyme activity induced by UV-B was significantly enhanced and the results show that the shikimate pathway enhances the can enhance the indole biosynthesis pathway, and ultimately laid the foundation for the synthesis of indole alkaloids. (2) sweetautumn Clematis in adenylate isopentenyltransferase the cloning and prokaryotic expression of the transcriptome sequencing, we obtained the EST fragment of adenylate isopentenyltransferase cone of Clematis in leaves, followed by rapid amplification of RT-PCR ends (rapid and cDNA amplification of cDNAends, RACE), the the nucleotide sequence of 1374 BP in length, including the length of ORF region of 332 aa. Then predicted and analyzed the sequence alignments and phylogenetic trees on physicochemical properties of the sequence encoding the CtIPT protein, the results showed that the protein is a molecular weight of 37.2 kDa hydrophilic protein exist adenylate isopentenyltransferase (ATP/GTP binding sequence feature sequence region region), and tomato S1IPT3/4 thaliana and AtIPT3/5/7 are very similar, indicating that the protein was adenylate isopentenyltransferase. Then, the influence of UV-B induction on CtIPT expression was detected by qRT-PCR. The results showed significant inhibition effect, indicating that CtIPT might have the function of resisting UV stress. Then to pET-28a (+), pGEX-4T-1 and pMAL-c2X plasmid expression vector of CtIPT was cloned, and the prokaryotic expression in Escherichia coli BL21, SDS-PAGE results showed that 180rpm at 16 DEG C, 0.5mM, IPTG induced expression conditions of 6 h can be successfully expressed the target protein, but only in BL21-pMAL-c2X-CtIPT (including MBP label) in soluble form. At last, the target protein adenylyl isoamyl transferase was obtained by Amylose resin affinity chromatography column.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S567.19

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